Stabilization of G-quadruplex structure on vascular endothelial growth factor gene promoter depends on CpG methylation site and cation type.

BACKGROUND: DNA methylation at the 5-position of cytosine is an epigenetic modification of CpG dinucleotides. In addition to CpG methylation, the G-quadruplex (G4) structure has been reported as a regulator of gene expression. The identification of G4 forming sequences in CpG islands suggests an involvement of CpG-methylated G4 structures in biological processes; however, few reports have addressed the effects of CpG methylation on G4 structure. METHODS: The thermostability of a methylated, 21-mer G4 structure located on the vascular endothelial growth factor (VEGF) gene promoter containing four CpG sites (C1, C6, C11, and C17) were investigated using circular dichroism (CD) spectral analysis. RESULTS: CD melting analysis revealed that VEGF G4 was stabilized by a single CpG methylation on C11 in the presence of Na+ and Mg2+. However, either C1 or C11 methylation enhanced VEGF G4 thermal stability in the presence of K+. CONCLUSIONS: Single CpG methylation appears to enhance VEGF G4 thermostability in a manner dependent on both the CpG methylation site and cation type. GENERAL SIGNIFICANCE: These results are expected to contribute to the elucidation of the roles of CpG methylation-stabilized G4 structures in biological processes.

A quantitative homogeneous assay for global DNA methylation levels using CpG-binding domain- and methyl-CpG-binding domain-fused luciferase.

Global DNA methylation levels have been considered as biomarkers for cancer diagnostics because transposable elements that constitute approximately 45% of the human genome are hypomethylated in cancer cells. We have previously reported a homogeneous assay for measuring methylated CpG content of genomic DNA based on bioluminescence resonance energy transfer (BRET) using methyl-CpG-binding domain (MBD)-fused luciferase (MBD-luciferase). In this study, a homogeneous assay for measuring unmethylated CpG content of genomic DNA in the same platform was developed using CXXC domain-fused luciferase (CXXC-luciferase) that specifically recognizes unmethylated CpG. In this assay, CXXC-luciferase recognizes unmethylated CpG on genomic DNA, whereby BRET between luciferase and the fluorescent DNA intercalating dye is detected. We demonstrated that the BRET signal depended on the genomic DNA concentration (R2 = 0.99) and unmethylated CpG content determined by the bisulfite method (R2 = 0.97). There was a significant negative correlation between the BRET signal of the CXXC-luciferase-based assay and that of the MBD-luciferase-based assay (R2 = 0.92). Moreover, we demonstrated that the global DNA methylation level determined using the bisulfite method was dependent on the ratio of the BRET signal in the MBD-luciferase-based assay to the total BRET signal in the MBD-luciferase- and CXXC-luciferase-based assays (R2 = 0.99, relative standard deviation < 2.2%, and analysis speed < 35 min). These results demonstrated that global DNA methylation levels can be quantified by calculating the BRET signal ratio without any calibration curve.

Global DNA Methylation Detection System Using MBD-Fused Luciferase Based on Bioluminescence Resonance Energy Transfer Assay.

DNA methylation plays an important role in the regulation of gene expression. In normal cells, transposable elements that constitute approximately 45% of the human genome are highly methylated to silence their expression. In cancer cells, transposable elements are hypomethylated; therefore, global DNA methylation level is considered as a biomarker for cancer diagnostics. In this study, a homogeneous assay for measuring global DNA methylation level based on bioluminescence resonance energy transfer (BRET) was developed using methyl-CpG binding domain (MBD)-fused luciferase. In this assay, the MBD-luciferase recognizes methylated CpG, thus, BRET between the luciferase and fluorescent DNA intercalating dye is detected. We demonstrated that the BRET signal depended on the DNA methylation level of the target DNA. Moreover, the BRET signal was correlated with the LINE1 DNA methylation level on human genomic DNA, as determined by the bisulfite method. These results indicate that the global DNA methylation level of human genomic DNA could be detected simply by measuring the BRET signal.

Detection of DNA Methylation of G-Quadruplex and i-Motif-Forming Sequences by Measuring the Initial Elongation Efficiency of Polymerase Chain Reaction.

DNA methylation has been proposed as one of the promising biomarkers for cancer diagnosis. In this study, we developed a DNA methylation detection system utilizing G-quadruplex and i-motif-forming sequences that requires neither sodium bisulfite treatment nor methylated DNA ligands. We hypothesized that G-quadruplex and i-motif structures would be stabilized by DNA methylation and arrest DNA polymerase activity during quantitative polymerase chain reaction (qPCR). The PCR products from VEGF, RET G-quadruplex, and i-motif-forming sequences were used as templates and analyzed by qPCR. Our results indicated that the initial elongation efficiency of PCR decreased with increasing DNA methylation levels in the G-quadruplex and i-motif-forming sequences. Moreover, we demonstrated that the initial elongation efficiency of PCR decreased with increased DNA methylation of the VEGF region on genomic DNA. These results indicated that DNA methylation of the G-quadruplex and i-motif-forming sequences on genomic DNA can be detected by qPCR.

Improving the efficiency of evolutionary de novo peptide design: strategies for probing configuration and parameter settings.

Evolutionary molecular design based on genetic algorithms (GAs) has been demonstrated to be a flexible and efficient optimization approach with potential for locating global optima. Its efficacy and efficiency are largely dependent on the operations and control parameters of the GAs. Accordingly, we have explored new operations and probed good parameter setting through simulations. The findings have been evaluated in a helical peptide design according to "Parameter setting by analogy" strategy; highly helical peptides have been successfully obtained with a population of only 16 peptides and 5 iterative cycles. The results indicate that new operations such as multi-step crossover-mutation are able to improve the explorative efficiency and to reduce the sensitivity to crossover and mutation rates (CR-MR). The efficiency of the peptide design has been furthermore improved by setting the GAs at the good CR-MR setting determined through simulation. These results suggest that probing the operations and parameter settings through simulation in combination with "Parameter setting by analogy" strategy provides an effective framework for improving the efficiency of the approach. Consequently, we conclude that this framework will be useful for contributing to practical peptide design, and gaining a better understanding of evolutionary molecular design.

Application of peptide probe for evaluating affinity properties of proteins using quartz crystal microbalance.

This study proved a possibility of a peptide probe for evaluating affinity properties of proteins. We have designed and synthesized three different peptide probes, H-Ala3-(Gly-Pro5)3-Gly-OH (peptide A), H-Ala3-(Gly-Pro5)-Gly-OH (peptide B) and H-Ala3-Gly-OH (peptide C) for testing their affinities to profilin. Each peptide probe was immobilized on a quartz crystal microbalance (QCM) sensor. The QCM sensor with the peptide A showed a 93 Hz decrease of resonant frequency which indicated profilin bound to the QCM sensor in a single layer. In a successive reaction with actin, the QCM analysis resulted in a 123 Hz decrease of resonant frequency which showed actin bound to the QCM sensor. A fluorescence microscope image of the sensor surface exhibited clear fluorescence after binding a rhodamine labeled actin on the sensor surface. These results supported stepwise reactions of profilin binding to the peptide A and actin binding to profilin. In the three peptide probes, the peptide A showed the highest affinity to profilin, i.e., sequence dependent affinity was confirmed.

Exploration of structural features of monomeric helical peptides designed with a genetic algorithm.

A genetic algorithm (GA)-based strategy to dissect the determinants of peptide folding into alpha-helix was developed. The structural information of helical peptides was obtained with respect to patterns of sequence variability. In many previously reported studies the intrinsic alpha-helical propensities of amino acids although sequence-dependent are apparently independent of the amino acid position. In this research, monomeric helical peptides selected from possible sequences produced by a GA-chemical synthesis were analyzed to identify possible influential structural features. These hexadeca-peptides were obtained after four successive generations. A total of 128 synthetic peptides were evaluated via circular dichroism (CD) measurements in aqueous solution, while the mean ellipticity at 222 nm confirmed the monomeric state of the peptides. The results presented here show that our GA-based strategy may be useful in the design of proteins with increased alpha-helix content.

A surface plasmon resonance probe with a novel integrated reference sensor surface.

A surface plasmon resonance (SPR) sensor probe with integrated reference surface is described. In order to fabricate the integrated reference surface, two dielectric layers with different thickness were deposited on the single gold SPR sensor surface via plasma polymerization of hexamethyldisiloxane. The working sensor surface was a 34 nm dielectric layer with immobilized bovine serum albumin (BSA) antigen and an adjacent thin 1 nm dielectric layer without BSA provided reference surface. A specific immunoreaction of anti-BSA antibody was detected after immersion of the SPR probe into sample solution. Simultaneous observation of reference and working surface response enabled determination of the immunoreaction without the need for the baseline measurement. Moreover, compensation of nonspecific adsorption could be confirmed using anti-human serum albumin antibody.

Cyanide hydrolysis in a cyanide-degrading bacterium, Pseudomonas stutzeri AK61, by cyanidase.

The cyanide-degrading bacterial strain AK61 was isolated from waste water at a metal-plating plant. The isolated strain was characterized by Gram-staining, quinone analysis, fatty acid profile and the API 20NE identification system, and identified as Pseudomonas stutzeri. Whole cells were able to degrade cyanide rapidly in a 1 mM solution containing no organic substances, and produced ammonia as a product. The induction of the cyanide-degrading activity of P. stutzeri AK61 did not depend on the presence of cyanide in the culture medium during growth. The cyanide-degrading enzyme was purified approximately 49-fold from a cell extract of P. stutzeri AK61. The enzyme had a K(m) of 1.7 mM for cyanide and a specific activity of 54.6 mumol ammonia produced min-1. The activity of the enzyme was optimal at 30 degrees C and pH 7.5. The results of SDS-PAGE, gel-filtration chromatography and NH2-terminal amino acid sequence analysis of the enzyme indicated that the functional enzyme was an aggregated protein consisting of a 38 kDa polypeptide. Like cyanidase (cyanide dihydratase), it was shown that the enzyme catalysed the hydrolysis of cyanide to ammonia and formate.